Protocols &Technical Information
Yeast Deletion Database (consortium members only)
Mating Test Protocol
Background:
We used BY4710 (Mat a trp-) and BY4 711 (Mat a trp-) as mating testers. Mating tests can also be performed with strains streaked out on plates. After checking strains by patching them out on their respective drop out plates, the strains can be crossed to a different auxotroph background strain to test for correct mating types on SD plates. Diploids should not mate with either A or alpha tester, therefore serve as negative control.
Day 1:
Transfer strains to be tested from 96-well microtiter plate (MTP) into a fresh MTP containing 50 to 75 ul YPD in each well using a 96-pin tool .
Streak out the a or a mating testers onto YPD plate so it makes a coarse lawn.
Incubate MTP and tester plates overnight at 30 °C.
Day 2:
Using sterile velvets and replicating block, transfer tester strain from YPD plate unto a maximum of 6 SD plates. Change velvet and repeat for the other tester strain using fresh SD plates.
Check the growth of the deletion strains in the MTP.
Transfer a small amount (less than 1 ul) of liquid culture from MTP onto A tester plate or alpha tester plate (sterilize the pin tips by rinsing in water first, followed by ethanol and flaming). Be sure to include proper controls on the SD plates.
Incubate at 30 °C.
Day 3 to 5:
Check for growth. Colonies are formed only after successful mating between opposite mating types and complementation of auxotrophy on SD plates.